TY - JOUR A1 - Bröker, Nina Kristin A1 - Gohlke, Ulrich A1 - Müller, Jürgen J. A1 - Uetrecht, Charlotte A1 - Heinemann, Udo A1 - Seckler, Robert A1 - Barbirz, Stefanie T1 - Single amino acid exchange in bacteriophage HK620 tailspike protein results in thousand-fold increase of its oligosaccharide affinity T2 - Glycobiology N2 - Bacteriophage HK620 recognizes and cleaves the O-antigen polysaccharide of Escherichia coli serogroup O18A1 with its tailspike protein (TSP). HK620TSP binds hexasaccharide fragments with low affinity, but single amino acid exchanges generated a set of high-affinity mutants with submicromolar dissociation constants. Isothermal titration calorimetry showed that only small amounts of heat were released upon complex formation via a large number of direct and solvent-mediated hydrogen bonds between carbohydrate and protein. At room temperature, association was both enthalpy- and entropy-driven emphasizing major solvent rearrangements upon complex formation. Crystal structure analysis showed identical protein and sugar conformers in the TSP complexes regardless of their hexasaccharide affinity. Only in one case, a TSP mutant bound a different hexasaccharide conformer. The extended sugar binding site could be dissected in two regions: first, a hydrophobic pocket at the reducing end with minor affinity contributions. Access to this site could be blocked by a single aspartate to asparagine exchange without major loss in hexasaccharide affinity. Second, a region where the specific exchange of glutamate for glutamine created a site for an additional water molecule. Side-chain rearrangements upon sugar binding led to desolvation and additional hydrogen bonding which define this region of the binding site as the high-affinity scaffold. KW - bacterial O-antigen KW - carbohydrate interaction KW - site-directed mutagenesis KW - structural thermodynamics KW - tailspike protein Y1 - 2013 UR - https://publishup.uni-potsdam.de/frontdoor/index/index/docId/35431 SN - 0959-6658 VL - 23 IS - 1 SP - 59 EP - 68 PB - Oxford Univ. Press CY - Cary ER -